Branched chain alpha-ketoacid dehydrogenase provides an excellent model by which to study the synthesis and assembly of mitochondrial multienzyme complexes. The proteins of this complex are encoded in the nucleus on separate genes and must assemble in a specific stoichiometry within the mitochondria. Inherited mutations in humans are known to affect the function of this complex. These mutations continue to be expressed in cells cultured from the patients and therefore can be used to study mutant and wild type gene expression and protein function. The plan of this project is to isolate cDNA and genomic clones for the various protein components. The nucleotide sequence for these DNA molecules will be determined and the wild type structures compared with mutant structures. This can easily be done using the polymerase chain reaction to amplify specific regions of the genes. Cells cultured from some individuals expressing defects in the branched chain dehydrogenase complex activity have been shown to lack one or another protein component of the complex. With clones for the missing component the cells can be transfected with the cloned DNA for the missing component and return of function can be determined. In this way we can begin to define the mutations at the gene level which give rise to Maple Syrup Urine disease.